Live Single-cell Mass Spectrometry Research Articles

Development of a highly efficient solubilization method for mass spectrometric analysis of phospholipids in living single cells.

Phospholipids are vital constituents of the cell membrane and aid in signal transduction. Phospholipid profiles vary distinctively with the cell type. Notably, specific phospholipid molecules are present in significantly higher or lower concentrations in cancer cells versus normal cells. In this study, live single-cell mass spectrometry (MS) was developed for analyzing phospholipids at the single-cell level. This method facilitates rapid molecular analysis of cells under microscopic observation. For nanoelectrospray ionization, phospholipids were extracted from single cells isolated in a glass capillary through a high-efficiency process. Cell-derived phosphatidylcholines were detected with high sensitivity when trehalose C14 was added as a solubilizing reagent. Trehalose C14 can solubilize cells at low concentrations owing to its low critical micelle concentration, and exerts minimal matrix effects (such as suppressing ionization and causing peak overlap) in the MS analysis of cellular molecules. Analyses of phospholipids in Raji and HEV0070 cells using the developed method revealed specific peaks of phosphatidylcholine and sphingomyelin in the respective cells. The developed technique not only affords phospholipid profiles at the single-cell level, but also holds promise for identifying biomarkers associated with various diseases, particularly cancer.

Analysis of the intracellular localization of amiodarone using live single-cell mass spectrometry

Amiodarone is a well-known antiarrhythmic drug with side effects including phospholipidosis. However, it is not clear how amiodarone and its metabolites are localized in the cell. In the present study, the localization of amiodarone in the cytosol, vacuoles, and lipid droplets of a single HepG2 human hepatocellular carcinoma cell was determined directly using live single-cell mass spectrometry. The cytosol, vacuoles, and lipid droplets of a single HepG2 cell treated with amiodarone were separately captured using a nano-spray tip under a fluorescence microscope after visualizing the lipid droplets using a fluorescent probe. This assay showed a linearity in the measurement of amiodarone levels with R2 values of 0.9996 and 0.9998 in the cell lysates and serum, respectively. The peak intensities of amiodarone and its metabolites in lipid droplets and vacuoles were significantly higher than those in the cytosol, while those in lipid droplets were higher than those in vacuoles. Amiodarone metabolites were detected in both lipid droplets and the cytosol. Live single-cell mass spectrometry combined with fluorescence imaging demonstrated clear localization of amiodarone and its metabolites in lipid droplets separately from the vacuole. This assay system combined with fluorescence imaging could be useful for investigating the intracellular localization of various drugs and their metabolites.

Single-Cell Native Mass Spectrometry of Human Erythrocytes.

Native mass spectrometry (MS) enables the determination of the molecular mass of protein complexes. Generally, samples for native MS are isolated, purified, and prepared in volatile solutions. However, to understand the function of proteins in living cells, it is essential to characterize the protein complex as is, without isolation/purification of the protein, using the smallest possible amount of the sample. In the present study, we modified the "live single-cell MS" method, which has mainly been used in metabolomics, and applied it to observe hemoglobin directly sampled from human erythrocytes. By optimizing the experimental methods and conditions, we obtained native mass spectra of hemoglobin using only a single erythrocyte, which was directly sampled into a nanoelectrospray ionization emitter using a micromanipulator and microinjector system. That is, our method enables the analysis of ∼0.45 fmol of hemoglobin directly sampled from an erythrocyte. To our knowledge, this is the first report of native MS for endogenous proteins using a single intact human cell.

Live Single-Cell Mass Spectrometry in Plant Science: Quantification of Plant Hormones and Future Prospects

Live Single-Cell Mass Spectrometry in Plant Science: Quantification of Plant Hormones and Future Prospects

Identification to species level of live single microalgal cells from plankton samples with matrix-free laser/desorption ionization mass spectrometry

IntroductionMarine planktonic communities are complex microbial consortia often dominated by microscopic algae. The taxonomic identification of individual phytoplankton cells usually relies on their morphology and demands expert knowledge. Recently, a live single-cell mass spectrometry (LSC-MS) pipeline was developed to generate metabolic profiles of microalgae.ObjectiveTaxonomic identification of diverse microalgal single cells from collection strains and plankton samples based on the metabolic fingerprints analyzed with matrix-free laser desorption/ionization high-resolution mass spectrometry.MethodsMatrix-free atmospheric pressure laser-desorption ionization mass spectrometry was performed to acquire single-cell mass spectra from collection strains and prior identified environmental isolates. The computational identification of microalgal species was performed by spectral pattern matching (SPM). Three similarity scores and a bootstrap-derived confidence score were evaluated in terms of their classification performance. The effects of high and low-mass resolutions on the classification success were evaluated.ResultsSeveral hundred single-cell mass spectra from nine genera and nine species of marine microalgae were obtained. SPM enabled the identification of single cells at the genus and species level with high accuracies. The receiver operating characteristic (ROC) curves indicated a good performance of the similarity measures but were outperformed by the bootstrap-derived confidence scores.ConclusionThis is the first study to solve taxonomic identification of microalgae based on the metabolic fingerprints of the individual cell using an SPM approach.

The complexity of intercellular localisation of alkaloids revealed by single-cell metabolomics.

Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C.roseus, cell-specific TIAs localisation and accumulation with 10μm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C.roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

Live Single-Cell Metabolomics With Matrix-Free Laser/Desorption Ionization Mass Spectrometry to Address Microalgal Physiology.

Unicellular phototrophic algae can form massive blooms with up to millions of individual cells per milliliter in freshwater and marine ecosystems. Despite the temporal dominance of bloom formers many algal species can co-exist and compete for nutrients and space, creating a complex and diverse community. While microscopy and single cell genomics can address the taxonomic inventory, the cellular metabolome has yet to be thoroughly explored to determine the physiological status of microalgae. This might, however, provide a key to understand the observed species diversity in the homogeneous environment. Here, we introduce an effective, rapid and versatile method to analyze living single cells from aqueous substrata with laser-desorption/ionization mass spectrometry (LDI-MS) using a simple and inexpensive matrix-free support. The cells deposited on a cultivation-medium wetted support are analyzed with minimal disturbance as they remain in their natural viable state until their disruption during LDI-MS. Metabolites desorbed from single cells are analyzed on High-Resolution Mass Spectrometry (HR-MS) using the Orbitrap FT-MS technology to fingerprint cellular chemistry. This live single-cell mass spectrometry (LSC-MS) allows assessing the physiological status and strain-specifics of different microalgae, including marine diatoms and freshwater chlorophytes, at the single-cell level. We further report a reliable and robust data treatment pipeline to perform multivariate statistics on the replicated LSC-MS data. Comparing single cell MS spectra from natural phytoplankton samples and from laboratory strains allows the identification and discrimination of inter and intra-specific metabolic variability and thereby has promising applications in addressing highly complex phytoplankton communities. Notably, the herein described matrix-free live-single-cell LDI-HR-MS approach enables monitoring dynamics of the plankton and might explain why key-players survive, thrive, avoid selective feeding or pathogenic virus and bacteria, while others are overcome and die.

Single-Cell Screening of Tamoxifen Abundance and Effect Using Mass Spectrometry and Raman-Spectroscopy.

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC-MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC-MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC-MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC-MS to investigate pharmacokinetics at the single-cell level.

Live single cell mass spectrometry reveals cancer-specific metabolic profiles of circulating tumor cells.

Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics‐based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type.

Live Single-Cell Mass Spectrometry (LSC-MS) for Plant Metabolomics.

Live single-cell mass spectrometry (LSC-MS) allows for the detection of hundreds to thousands of metabolite peaks acquired from a single plant cell within a few minutes. Plant cells are first observed under a stereomicroscope, a cell of interest is chosen, and then sampled using a metal-coated glass microcapillary for subsequent analysis. A few microliters of ionization solvent is then added to the rear end of the capillary followed by the introduction of the capillary's content directly into the mass spectrometer. High voltage is applied between the capillary and the mass spectrometer inlet to induce nanospray ionization. Metabolite structural confirmation is performed using tandem mass spectrometry analysis (MS/MS) and fragments are matched with MS/MS databases to predict metabolic pathways. This method enables swift and direct molecular detection and identification of specific metabolites from a single plant cell along with their localization within the cell, which will allow for comprehensive understanding of plant metabolomics on a single cell level.

升島努先生を偲んで―1細胞質量分析までの研究の足跡―

升島努先生を偲んで―1細胞質量分析までの研究の足跡―

Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS

Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue.

Quantitative Live Single-cell Mass Spectrometry with Spatial Evaluation by Three-Dimensional Holographic and Tomographic Laser Microscopy

The locations and volumes of the contents of a single HepG2 cell were visualized under three-dimensional (3D) holographic and tomographic (HT) laser microscopy, colored by refractive index, not staining. After trapping the specific area of a target cell in a nanospray tip, quantification was performed by live single-cell mass spectrometry. Comparison of the HepG2 cells' before and after 3D-HT images allowed the inference of the precise volume and original location of the trapped cell contents. The total amount of a trapped molecule was estimated. The images also revealed morphological changes in the cell structure caused by the manipulation.

Direct Lipido-Metabolomics of Single Floating Cells for Analysis of Circulating Tumor Cells by Live Single-cell Mass Spectrometry.

Direct trapping of a single floating cell, i.e. a white blood cell from a drop of blood, within a nanospray tip was followed by super-sonication after the addition of ionization solvent. Molecular detection of an increased number of peaks with a higher intensity and a wider m/z range, which extends from metabolites to lipids, was acquired than of that without sonication. This method was applied to a few separated circulating tumor cells (CTC) from a neuroblastoma patient's blood to obtain their lipido-metabolomic molecular profile at the single cell level. In addition to vital molecules such as amino acids, catechol amine metabolites, which are specific to neuroblastoma, and drugs included in the patient's course of therapy were detected. This established "direct single-cell lipido-metabolomic method" seems to be useful for direct and wide range molecular detection not only for many live single-cells, but also for rare cells, such as CTCs, for future molecular diagnosis.

Fluorescence Probing Live Single-cell Mass Spectrometry for Direct Analysis of Organelle Metabolism.

Mitochondria in a live HepG2 cell were visualized with a fluorescent probe to specify their location and state in a living cell. Then, mitochondria were selectively captured with a nanospray tip under fluorescence microscope, and thousands of small molecular peaks were revealed and unique steroids specific to mitochondria were also found. This fluorescence imaging combined with live single-cell mass spectrometry opens the door to the analysis of site- and state-specific molecular detection to elucidate precise molecular mechanisms at the single-cell and organelle level.

Direct metabolomics for plant cells by live single-cell mass spectrometry.

Live single-cell mass spectrometry (live MS) provides a mass spectrum that shows thousands of metabolite peaks from a single live plant cell within minutes. By using an optical microscope, a cell is chosen for analysis and a metal-coated nanospray microcapillary tip is used to remove the cell's contents. After adding a microliter of ionization solvent to the opposite end of the tip, the trapped contents are directly fed into the mass spectrometer by applying a high voltage between the tip and the inlet port of the spectrometer to induce nanospray ionization. Proteins are not detected because of insufficient sensitivity. Metabolite peaks are identified by exact mass or tandem mass spectrometry (MS/MS) analysis, and isomers can be separated by combining live MS with ion-mobility separation. By using this approach, spectra can be acquired in 10 min. In combination with metabolic maps and/or molecular databases, the data can be annotated into metabolic pathways; the data analysis takes 30 min to 4 h, depending on the MS/MS data availability from databases. This method enables the analysis of a number of metabolites from a single cell with rapid sampling at sub-attomolar-level sensitivity.

"MS-Patch-Clamp" or the Possibility of Mass Spectrometry Hybridization with Patch-Clamp Setups for Single Cell Metabolomics and Channelomics

In this projecting work we propose a mass spectrometric patch-clamp equipment with the capillary performing both a local potential registration at the cell membrane and the analyte suction simultaneously. This paper provides a current literature analysis comparing the possibilities of the novel approach proposed with the known methods, such as scanning patch-clamp, scanning ion conductance microscopy, patch clamp based on scanning probe microscopy technology, quantitative subcellular secondary ion mass spectrometry or "ion microscopy", live single-cell mass spectrometry, in situ cell-by-cell imaging, single-cell video-mass spectrometry, etc. We also consider the ways to improve the informativeness of these methods and particularly emphasize the trend at the increasing of the analysis complexity. We propose here the way to improve the efficiency of the cell trapping to the capillary during MS-path-clamp, as well as to provide laser surface ionization using laser trapping and tweezing of cells with the laser beam transmitted through the capillary as a waveguide. It is also possible to combine the above system with the microcolumn separation system or capillary electrophoresis as an optional direction of further development of the complex of analytical techniques emerging from the MS variation of patch-clamp.

Direct molecular analysis of plant cells by live single-cell mass spectrometry

Direct molecular analysis of plant cells by live single-cell mass spectrometry

Drug Metabolite Heterogeneity in Cultured Single Cells Profiled by Pico-Trapping Direct Mass Spectrometry

We investigated the heterogeneity of tafluprost metabolism in primary human hepatocytes at a single-cell level by live single-cell mass spectrometry (MS). Picoliter volumes of cytoplasm were analyzed by nano-electrospray ionization MS in order to obtain single-cell metabolite profiles. The subcellular components of a single tafluprost-treated human hepatocyte were isolated and the single-cell metabolite profile was compared with those of traditional bulk hepatocyte analysis. In the bulk hepatocyte analysis, liquid chromatography-MS showed the averaged metabolism of tafluprost to tafluprost acid (TA) and β-oxidized metabolites. However, live single-cell MS showed that tafluprost metabolism varied among individual cells. In addition, there was significant variation in the quantities of TA and a major metabolite, dinor-TA, among cells, whereas there was no significant variation in 7-ethoxycoumarin metabolism. Thus, live single-cell MS successfully detected the heterogeneity of drug metabolism in individual living hepatocytes.

Molecular and Functional Analysis of Cellular Phenomena Using Single-Cell Mass Spectrometry

Single-cell analysis has attracted attention in many fields of biological studies as a tool to survey the precise mechanisms of cellular and molecular behavior. The development of sensitive mass spectrometry allows the study of molecules in single cells or small regions. Matrix-assisted laser desorption/ionization imaging mass spectrometry and secondary-ion mass spectrometry use in situ ionization of specimens on sample plates to visualize molecular distributions as images from mass spectra. Several single-cell mass spectrometry technologies that initially recover a single cell followed by ionization have been developed. Among them, only nanospray-mediated sampling and ionization named Live Single-cell Mass Spectrometry can be used for real-time analysis. This paper explains that method in detail.